Dai Xufang, Yan Xiaojing, Xie Peng, Lian Jiqin
Chongqing Key Laboratory of Psychological Diagnosis and Educational Technology for Children with Special Needs.
Facultiy of Educationfor Children with Special Needs, College of Education Science, Chongqing Normal University, Chongqing 400047, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2018 Dec 30;38(12):1415-1420. doi: 10.12122/j.issn.1673-4254.2018.12.03.
To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.
Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.
VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.
VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.
研究丙戊酸钠（VPA）对SH-SY5Y细胞中miR-34c-5p/ATG4B信号通路激活及自噬的影响。
将常规培养的SH-SY5Y细胞用不同剂量的VPA处理24小时，分别采用qRT-PCR和免疫印迹法评估ATG4B和miR-34c-5p的mRNA水平变化以及ATG4B的蛋白表达。使用报告基因测定法评估转染含ATG4B启动子的质粒对VPA处理的SH-SY5Y细胞中ATG4B启动子活性的影响。在单独用VPA或VPA联合转录抑制剂放线菌素D处理的SH-SY5Y细胞中，用qPCR分析ATG4B mRNA的稳定性。在单独用VPA或VPA联合miR-34c-5p模拟物或拮抗剂处理的SH-SY5Y细胞中，用qPCR检测miR-34c-5p的表达水平，并评估miR-34c-5p在VPA诱导的ATG4B下调中的作用。通过检测VPA或VPA联合miR-34c-5p拮抗剂处理后细胞中LC3-Ⅱ的表达来评估自噬水平的变化。
VPA剂量依赖性地降低SH-SY5Y细胞中ATG4B在mRNA和蛋白水平的表达。VPA处理对ATG4B的启动子活性无显著影响，但明显降低了SH-SY5Y细胞中ATG4B的mRNA稳定性。VPA处理上调了miR-34c-5p的表达，并且miR-34c-5p拮抗剂逆转了VPA诱导的SH-SY5Y细胞中ATG4B的下调。VPA还下调了SH-SY5Y细胞中LC3-Ⅱ的表达水平。
VPA可能通过激活miR-34c-5p/ATG4B信号通路抑制SH-SY5Y细胞中的自噬。
